Cigarette smoking is the number one cause of preventable death in the U.S. however, despite the known health consequences of smoking, 1 in 5 Americans continues to smoke. Most smokers desire to quit but success rates are low. Women have greater difficulty quitting smoking and suffer greater smoking-related health consequences than men. Greater knowledge of relapse vulnerabilities in women could help to identify treatment targets and result in interventions that improve health and save lives. The guiding hypothesis for this proposal is that fluctuations in gonadal sex hormones [estradiol (E) and progesterone (P)] over the course of the menstrual cycle (MC) may greatly impact relapse vulnerability through their actions on a powerful relapse motivator, smoking-related cue (SC) exposure. This hypothesis arises from animal studies showing that gonadal sex hormones dramatically affect reward systems and reward-related behavior. Specifically, E elevates ventral striatal dopamine and accelerates both drug-cued and drug-primed reinstatement, while P has opposite effects on drug-seeking behavior. Correspondingly, when women have high E levels, such as those occurring during the late follicular phase of the MC, they may find drugs more rewarding and drug-related cues more motivating, which could markedly undermine women's success in quitting smoking at that time. Our preliminary neuroimaging results show enhanced responses to SCs in the medial orbitofrontal cortex, a region known to be involved in the coding of reward magnitude, in women during the follicular compared to the luteal phase, but the direct effects of the hormonal milieu on neural responses to SCs remains untested, divesting us of critical, treatment-relevant information. Thus, the overarching goal of this proposal is to characterize the influence of the natural hormonal milieu on brain and behavioral responses to SCs. Specifically, in AIM 1, to link hormonal status to the brain responses to SCs, over the course of 3 monthly MCs, in a repeated measures counter- balanced design, we will compare the brain responses to highly appetitive SCs in a group of healthy, naturally- cycling females who are chronic smokers at 3 distinct time points within their natural MC. Given that the early follicular phase is associated with extremely low levels of both E and P, it offers a natural and ideal comparator condition. Sessions will be carefully timed to occur during high E, which corresponds to the late follicular phase, during high P, which corresponds to the mid-luteal phase, and during the time of the MC when both E and P are low (LEP). E/P concentrations will be used to verify conditions and as covariates to examine their associations with brain responses. In AIM 2, to link hormonal status and brain responses to the behavioral biases to SCs, in synchrony with the imaging sessions, women from Aim 1 will also participate in 3 laboratory sessions consisting of a neuro-behavioral battery that includes tasks to probe affective and attentional biases to SCs at both time points. This research addresses a critical gap in our knowledge: namely, the impact of the hormonal milieu on brain response to SCs, an important relapse trigger. The results may guide hormonally- informed treatment strategies with the potential to reduce relapse, thus improving health and saving lives.